Clin Exp Metastasis (2012) 29:207–216 DOI 10.1007/s10585-011-9443-3
RESEARCH PAPER
Erlotinib prevents experimental metastases of human small cell lung cancer cells with no epidermal growth factor receptor expression Adel Gomaa Mohammed Gabr • Hisatsugu Goto • Masaki Hanibuchi • Hirohisa Ogawa • Takuya Kuramoto • Minako Suzuki • Atsuro Saijo • Soji Kakiuchi • Van The Trung • Satoshi Sakaguchi • Yoichiro Moriya • Saburo Sone • Yasuhiko Nishioka Received: 2 September 2011 / Accepted: 6 December 2011 / Published online: 15 December 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs) show dramatic antitumor activity in a subset of patients with non-small cell lung cancer who have an active mutation in the epidermal growth factor receptor (EGFR) gene. On the other hand, some lung cancer patients with wild type EGFR also respond to EGFR–TKIs, suggesting that EGFR–TKIs have an effect on host cells as well as tumor cells. However, the effect of EGFR–TKIs on host microenvironments is largely unknown. A multiple organ metastasis model was previously established in natural killer cell-depleted severe combined immunodeficient mice using human lung cancer cells. This model was used to investigate the therapeutic efficacy of erlotinib, an EGFR–TKI, on multiple organ
metastases induced by human small cell lung cancer cells (SBC-5 cells) that did not express EGFR. Although erlotinib did not have any effect on the proliferation of SBC5 cells in vitro, it significantly suppressed bone and lung metastases in vivo, but not liver metastases. An immunohistochemical analysis revealed that, erlotinib significantly suppressed the number of osteoclasts in bone metastases, whereas no difference was seen in microvessel density. Moreover, erlotinib inhibited EGF-induced receptor activator of nuclear factor kappa-B expression in an osteoblastic cell line (MC3T3-E1 cells). These results strongly suggested that erlotinib prevented bone metastases by affecting host microenvironments irrespective of its direct effect on tumor cells. Keywords Erlotinib Bone metastasis Lung cancer Host microenvironment
A. G. M. Gabr T. Kuramoto S. Kakiuchi V. T. Trung S. Sone Department of Medical Oncology, Institute of Health Biosciences, The University of Tokushima Gradate School, Tokushima, Japan H. Goto M. Hanibuchi M. Suzuki A. Saijo S. Sakaguchi S. Sone Y. Nishioka (&) Department of Respiratory Medicine and Rheumatology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan e-mail:
[email protected] H. Ogawa Department of Molecular and Environmental Pathology, Institute of Health Biosciences, The University of Tokushima Gradate School, Tokushima, Japan Y. Moriya Chugai Pharmaceutical Co., Ltd., Tokyo, Japan
Abbreviations EGFR–TKI Epidermal growth factor receptor–tyrosine kinase inhibitor NSCLC Non-small cell lung cancer NK Natural killer SCID Severe combined immunodeficient SCLC Small cell lung cancer PTHrP Parathyroid hormone-related protein EGFR Epidermal growth factor receptor HUVECs Human umbilical vein endothelial cells IL Interleukin Ab Antibody VEGF Vascular endothelial growth factor MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium BMMSCs Bone marrow mesenchymal stem cells HGF Hepatocyte growth factor
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Introduction Lung cancer is the major cause of malignancy-related death worldwide. The mortality rate is 80–90%, which makes this disease the leading cause of cancer-related death [1]. The high mortality of this disease is primarily due to the difficulty of early diagnosis and the highly metastatic potential. Approximately 70% of lung cancer patients have already developed metastases to multiple organs by the time of the diagnosis [2]. There is no curative therapy for the metastases, and clinical management is palliative in many cases. Therefore, it is crucial to prevent and treat lung cancer metastases. Although the outcome of conventional chemotherapy for patients with advanced lung cancer is still unsatisfactory, recent studies have enabled the development of molecular targeting agents such as epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs). Treatment with the reversible EGFR–TKIs (gefitinib and erlotinib) results in dramatic antitumor activity in a subset of patients with non-small cell lung cancer (NSCLC). Approximately 75% of patients with epidermal growth factor receptor (EGFR) mutations respond to EGFR–TKIs [3, 4]. The inhibition of EGFR tyrosine kinase results in the induction of substantial apoptosis in these tumor cells because lung cancer cells that have an active mutation in the EGFR gene become addicted to the EGFR signaling pathway for their growth. This mechanism allows EGFR– TKIs to exert anti-tumor activity in NSCLC patients with an active mutation in the EGFR gene. However, it is noteworthy that an objective response of about 10% is also achieved with erlotinib treatment in NSCLC patients with wild type EGFR [5]. These clinical observations indicate that EGFR–TKIs might have other mechanisms of action in addition to their direct effect on tumor cells. Normanno et al. [6] demonstrated that gefitinib inhibits the recruitment of osteoclasts in bone lesions, by affecting the ability of bone marrow stromal cells to induce osteoclast differentiation and activation. Moreover, Cerniglia et al. [7] reported that erlotinib treatment of tumor-bearing mice alters vessel morphology and decreases vessel permeability in tumor xenografts. These observations suggest that EGFR–TKIs have the potential to modulate and affect host cells, but their effect on the host microenvironment in different organs with cancer metastases is largely unknown. The establishment of clinically relevant experimental metastasis models is crucial to understand the pathogenesis of lung cancer and its relationship to host microenvironment. A reproducible model of multiple organ metastases by human lung cancer cells was previously established in natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice [8, 9]. This model was used to elucidate the mechanisms of interactions of metastatic lung
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cancer cells with host organ microenvironment and the heterogeneity in organ microenvironments on the metastases of human lung cancer [10–14]. The present study sought to elucidate the action of EGFR–TKIs on host microenvironments in organs with lung cancer metastases. The study investigated the therapeutic efficacy of erlotinib, an EGFR–TKI, on multiple organ metastases induced by SBC-5 human small cell lung cancer (SCLC) cells that did not express EGFR in NK celldepleted SCID mice.
Materials and methods Cell cultures The human SCLC cell line, SBC-5 [15] was kindly provided by Drs. M. Tanimoto and K. Kiura (Okayama University, Okayama, Japan). The PC-9 human adenocarcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA). SBC-5 cells were maintained in Eagle’s MEM (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Grand Island, NY), penicillin (100 U/ml), and streptomycin (50 lg/ml). PC-9 cells were maintained in RPMI1640 (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% heat-inactivated FBS, penicillin and streptomycin. The MC3T3-E1 murine osteoblastic cell line subclone 4 was kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). MC3T3-E1 cells were maintained as preosteoblasts in a-MEM growth medium supplemented with 10% FBS, penicillin and streptomycin. The growth medium was supplemented with L-ascorbic acid (50 lg/ml) to induce differentiation, and the cells were cultured for 7 days [16]. All cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Reagents An anti-mouse interleukin (IL)-2 receptor b chain monoclonal antibody (Ab), TM-b1 (IgG2b), was kindly provided by Drs. M. Miyasaka and T. Tanaka (Osaka University, Osaka, Japan) [17]. Recombinant murine EGF was purchased from Invitrogen (Carlsbad, CA). None of these materials contained endotoxins, as determined by the limulus amebocyte assay (Seikagaku Kogyo, Tokyo, Japan: minimum detection level, 0.1 ng/ml). In vitro cell proliferation and migration assay Cell proliferation was determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) dye reduction method [18]. The human lung cancer cells
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(2 9 103 cells/100 ll) were plated into each well of a 96-well plate, and incubated overnight. Various concentrations of erlotinib were added and the cells incubated for 72 h. Fifty microliters of stock MTT solution (2 mg/ml; Sigma-Aldrich, St. Louis, MO) was added to all wells, and the cells were then further incubated for 2 h at 37°C. The media containing MTT solution were removed, and 100 ll of DMSO (Sigma-Aldrich, St. Louis, MO) was added. The absorbance was measured with an MTP-32 Microplate Reader (Corona Electric, Ibaragi, Japan) at test and reference wavelengths of 550 and 630 nm, respectively. Cell migration was determined using double chamber method. RPMI-1640 medium (0.75 ml) containing 10% FBS was applied to the lower chamber as chemoattractant, and SBC5 cells (5 9 104) in 0.5 ml of serum-free RPMI-1640 medium were seeded in inner chamber (8 lm-pore, BD Labware, Franklin Lakes, NJ) with the presence of 0, 0.01, 0.1 or 1 lM erlotinib, and incubated for 19 h at 37°C. After incubation, cells remaining on the upper side of the insert were removed, and the cells that migrated to the lower surface of the insert were counted in four different random fields at 1009 magnification. Each treatment was performed in triplicate. Animals Male, 6 to 8-week-old S EB-17/Icr-scid mice were obtained from Clea Japan (Osaka, Japan) and maintained under specific pathogen-free conditions throughout the experiment. The experimental protocol was reviewed and approved by the animal care and use committee of The University of Tokushima, and were performed according to their guidelines. Experimental metastasis with SBC-5 cells and the effect of erlotinib NK cells were depleted in SCID mice to facilitate the metastasis of human lung cancer cell lines. TM-b1 Ab (0.3 mg/mouse) was injected i.p. into SCID mice 2 days before tumor inoculation [8]. Tumor cells were harvested and washed with Ca2?- and Mg2?-free phosphate buffered saline (Nissui Pharmaceutical Co., Tokyo, Japan). Cell viability was determined by a trypan blue exclusion test and only single cell suspensions of [90% viability were used. SBC-5 cells (1 9 106 cells/0.3 ml/mouse) were inoculated into the lateral tail vein of unanesthetized SCID mice pretreated with TM-b1 Ab on day 0. Oral administration of erlotinib (10 or 30 mg/kg) was performed once daily from day 7 to 28. Vehicle was administered to the control group. Mice were anesthetized and humanely sacrificed on day 29 by cutting the subclavian artery, and all major organs were
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removed. Whole body X-ray photographs (Fuji Film, Tokyo, Japan) of tumor-bearing mice were taken just before sacrifice and bone metastases were independently evaluated on X-ray photographs by two authors [9]. The lungs were fixed in Bouin’s solution (Sigma-Aldrich, St. Louis, MO) for 24 h. The number of metastatic foci on the lungs, liver were counted macroscopically. Immunohistochemistry The hind limbs of the mice were taken and fixed in 10% formalin. The bone specimens were decalcified in 10% EDTA solution for 1 week and then embedded in paraffin. The paraffin-embedded tissue samples were cut into 3-lm sections and picked up on slides. Tartrate-resistant acid phosphatase (TRAP) staining was performed using a Sigma Diagnostics Acid Phosphatase Kit (Sigma Diagnostics, St. Louis, MO) for the detection of osteoclasts. The number of TRAP-positive osteoclasts at the tumor-bone interface in each slide was counted under a microscope in five random fields at 2009 magnification. Formalin fixed, paraffin embedded sections were stained with anti-mouse CD31 Ab (Santa Cruz Biotechnology, Santa Cruz, CA). CD31 positive microvessels were counted from five independent fields at 2009 magnification of each section. The sections were also stained with H&E for routine histologic examination. Western blot analysis The cells were seeded at 1 9 106 cells/dish in corresponding differentiation conditions to assess the effects of exogenous stimuli on the activation of the EGFR in MC3T3-E1 cells. Differentiated cells were cultured for 24 h in medium containing 1% FBS, and subsequently treated with recombinant EGF for 10 min in the absence or presence of various concentrations of erlotinib. Cells were lysed in M-PER (Pierce, Rockford, IL) containing phosphatase inhibitor cocktail and proteinase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and the protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA). Aliquots of 400 lg of total protein were immunoprecipitated with the anti-mouse EGFR Ab (Cell signaling, Danvers, MA), and the immune complexes were recovered with Protein G-Sepharose beads (GE Healthcare, Buckinghamshire, UK). Proteins were separated by SDS-PAGE (Invitrogen, Carlsbad CA) and then transferred to PVDF membranes (Atto, Tokyo, Japan). Washed membranes were incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, then incubated 1 h at room temperature with anti-phosphotyrosine Ab (1:1,000 dilution, cell signaling) or anti-EGFR Ab (1:2,00 dilution, cell signaling). The membranes were
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Reverse transcription polymerase chain reaction MC3T3-E1 cells were seeded at 1 9 106 cells/well in 6-well plates in the corresponding differentiation conditions. The differentiated cells were treated with recombinant EGF in the absence or presence of indicated dose of erlotinib for 48 h. Total cellular RNA was isolated by using RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols. RNA (0.5–2.0 lg) was reverse transcribed using a TM TaqManÒ RNA-to-CT 2-Step kit (Applied biosystems, Foster City, CA). Messenger The expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and EGFR mRNA were measured by real-time polymerase chain reaction (PCR) using Assay on Demand Taqman Gene expression primers and probes (Applied biosystems). PCR reaction conditions were those recommended by the manufacturer. Fluorescence signals were monitored after each PCR cycle with an ABI Prism 7700 sequence detection system (Applied Biosystems). CT values (cycle number where fluorescence exceeded a fixed threshold) were obtained for each target probe and normalized with the corresponding CT values for the internal control (mouse b2 microglobulin). The RNA levels were expressed as relative units. Statistical analysis The Mann–Whitney U test was used to evaluate the differences in the numbers of metastases into multiple organs (bone, liver, and lung) between the erlotinib-treated groups and the control (vehicle treated group), and to evaluate the differences in immunohistochemistry. The differences in in vitro experiments were analyzed by Student’s t test (twotailed). A P value of\0.05 was considered to be significant in all experiments.
Results Erlotinib did not affect the proliferation and migration of human SCLC, SBC-5 cells in vitro We have previously reported that SBC-5 cells do not express EGFR [12]. In this study, we first examined the effect of erlotinib on in vitro proliferation and migration of SBC-5 cells. As shown in Fig. 1a, clinically relevant doses
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A 120 100 80
%Growth
incubated for 30 min at room temperature with speciesspecific horseradish peroxidase conjugated secondary antibodies, and the immunoreactive bands were visualized using an enhanced chemiluminescent substrate (Pierce, Rockford, IL).
Clin Exp Metastasis (2012) 29:207–216
60 40 20 0
0
0.001
0.003
0.01
0.03
0.1
0.3
1
3
Erlotinib (µM)
B
Fig. 1 Effect of erlotinib on the proliferation and migration of human SCLC, SBC-5 cells in vitro. a SBC-5 cells (1 9 103 cells/well; open circles) or PC-9 cells (1 9 103 cells/well; closed circles) were plated into each well of a 96-well plate and incubated overnight. Various concentrations of erlotinib were added and the cells were incubated for 72 h. Cell growth was determined by MTT assay as described in ‘‘Materials and methods’’ section. Bars show SDs of the means of triplicate cultures. Data are representative of five independent experiments. b Cell migration of SBC-5 cells was assessed as described in ‘‘Materials and methods’’ section. Bars show SDs of the means of triplicate cultures. Data are representative of two independent experiments
(3 lM or less) of erlotinib [19] had no effect on the proliferation of SBC-5 cells. On the contrary, erlotinib significantly inhibited the proliferation of PC-9 cells, which harbor a deletion mutation on exon 19 of the EGFR gene, in a dose-dependent fashion (Fig. 1a), consistent with a previous report [20]. Cell migration of SBC-5 cells was also not affected by erlotinib treatment (Fig. 1b). Treatment with erlotinib inhibited bone and lung metastases by SBC-5 cells in NK cell-depleted SCID mice A multiple organ metastasis model with SBC-5 cells was established in SCID mice depleted of NK cells [9]. Intravenously inoculated SBC-5 cells (1 9 106/mouse)
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Effect of erlotinib on tumor angiogenesis
developed metastatic colonies in the lungs, liver, and bones (osteolytic metastasis) of NK cell-depleted SCID mice. This model was used to assess the therapeutic efficacy of erlotinib. Oral administration of erlotinib (30 mg/kg) from day 7 to 28 significantly inhibited the formation of osteolytic bone metastases (Fig. 2). Interestingly, treatment with erlotinib also inhibited distant metastases in the lungs, although it had no effect on liver metastases (Table 1). Erlotinib treatment was well tolerated and no obvious adverse events, such as body weight loss, were observed even in the 30 mg/kg group (data not shown). These in vivo results suggested that erlotinib prevented distant metastases by affecting the host microenvironments because erlotinib had no direct effect on the proliferation of SBC-5 cells in vitro.
Guillamo et al. [21] reported that EGFR–TKI (gefitinib) inhibits angiogenesis in a mouse experimental glioma model. Moreover, Riedel et al. [22] reported that addition of conditioned medium from EGFR antisense-treated tumor cells decreases endothelial cell migration and proliferation. These findings suggested that the prevention of SBC-5-induced metastasis formation by erlotinib could be due to the inhibition of angiogenesis. However, immunohistochemical staining with CD31 revealed no significant difference in the microvessel density in the lung, bone, and liver (Fig. 3). The effect of erlotinib on the proliferation of human umbilical vein endothelial cells (HUVECs) was also evaluated in vitro. Up to 3 lM of erlotinib did not
Fig. 2 Therapeutic efficacy of erlotinib against osteolytic bone metastases produced by human SCLC, SBC-5 cells. SBC-5 cells (1 9 106 cells/mouse) were inoculated into the tail vein of NK celldepleted SCID mice. Oral administration of erlotinib was performed
as described in ‘‘Materials and methods’’ section. Mice were sacrificed and bone metastases were determined radiographically on day 29. The representative pictures of vehicle- and erlotinib-treated mice are shown. The arrows indicate osteolytic bone metastases
Table 1 Effect of erlotinib on multiple organ metastasis produced by SBC-5 cells Treatment
Bone
Liver
Number of metastasis
Weight (g)
Number of metastasis
Weight (g)
Number of metastasis
Inc.
Med.
Inc.
Med.
Inc.
Med.
Range
Lung
Range
Med.
Range
Range
Med.
Range
Experiment 1 Vehicle
6/6
6
5–7
1.6
1.3–1.9
6/6
39
25–80
0.3
0.3–0.4
6/6
38
30–55
10 mg/kg 30 mg/kg
5/5 5/5
5 3*
3–6 2–5
1.4 2.0
1.0–1.7 1.8–3.4
5/5 5/5
23 26
12–35 2–38
0.3 0.3
0.2–0.5 0.3–0.4
5/5 5/5
20 15*
8–30 6–35
Vehicle
6/6
9
7–11
1.8
1.6–2.4
6/6
60
20–150
0.22
0.24–0.41
6/6
14
5–20
10 mg/kg
6/6
8
5–11
1.6
1.3–1.8
6/6
31
10–50
0.24
0.29–0.4
6/6
10
9–11
30 mg/kg
6/6
4*
2–6
1.5
1.8–3.3
6/6
24
12–40
0.21
0.3–0.4
6/6
7*
2–11
Experiment 2
6
SBC-5 cells (1 9 10 cells/mouse) were inoculated i.v. into NK cell-depleted SCID mice on day 0. Erlotinib (or vehicle) was given orally from day 7 to 28 after tumor cell inoculation. The metastatic profile was evaluated on day 29. Mann–Whitney U test was used to determine the significance of differences Inc. incidence, Med. median * Statistically significant difference compared with vehicle-treated group (P \ 0.05)
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A
Bone
Lung
Erlotinib
Control
Liver
Liver
B
Bone
Lung
CD31 positive cells (field)
20
15
10
5
0
Control
Erlotinib
Control
Erlotinib
Control
Erlotinib
Fig. 3 Effect of erlotinib on tumor angiogenesis. SBC-5 cells (1 9 106 cells/mouse) were inoculated into the tail vein of NK celldepleted SCID mice. Oral administration of erlotinib was performed as described in ‘‘Materials and methods’’ section. Each organ was collected on day 29, paraffin embedded, and sections were subjected
to immunohistochemical staining of CD31. a Representative pictures of immunohistochemical staining are shown (magnification, 9200). b CD31 positive microvessels were counted from five independent fields of each section. Bars show SEM of the means for five mice per group
affect the proliferation of HUVECs irrespective of the stimulation with recombinant human vascular endothelial growth factor (VEGF) (data not shown).
treatment significantly reduced the number of osteoclasts (TRAP positive cells) in metastatic bone lesions.
Erlotinib reduced the number of osteoclast in bone metastasis lesion A previous study demonstrated the importance of osteoclasts in bone metastasis development by SBC-5 cells [9, 10]. Immunohistochemical staining (TRAP staining) was performed to evaluate the number of osteoclasts in metastatic bone lesions. Figure 4 demonstrates that erlotinib
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Erlotinib inhibited EGF-induced RANKL expression in osteoblastic cell line MC3T3-E1 cells RANKL is one of the key molecules responsible for the formation of osteolytic bone metastasis in various types of cancer [23]. RANKL expressed by osteoblasts activates osteoclasts by binding to its receptor (RANK). The fact that the number of osteoclasts in bone metastasis lesions decreased after erlotinib treatment suggested that erlotinib
Clin Exp Metastasis (2012) 29:207–216
A
Control
213
Erlotinib
A p-EGFR EGFR EGF
-
-
-
+
+
+
µM) Erlotinib (µ
0
1
0.1
0
1
0.1
B
TRAP positive cells (/field)
B
30
20
*
10
0
EGF
-
- -
-
-
+ + + + +
Erlotinib (µM)
0
0.125 0.25
0.5
1
0
0.125 0.25 0.5
1
C Control
Erlotinib
Fig. 4 The effect of erlotinib on osteoclast recruitment in metastatic bone lesion. SBC-5 cells (1 9 106 cells/mouse) were inoculated into the tail vein of NK cell-depleted SCID mice. Oral administration of erlotinib was performed as described in ‘‘Materials and methods’’ section. Metastatic bone lesions were collected on day 29, and sections were subjected to TRAP staining to evaluate osteoclast recruitment. a Representative pictures of TRAP staining of bone are shown (magnification, 9200). TRAP positive cells were stained in purple. b The number of osteoclasts (TRAP positive cells) was counted from five independent fields of each section. Bars show the SEM of the means for five mice per group. *P \ 0.01
suppresses osteoclast differentiation and activation by inhibiting the RANK–RANKL axis. The expression of EGFR in the osteoblastic cell line MC3T3-E1 cells was evaluated, since EGFR is reported to be expressed in osteoblasts but not in osteoclasts [16]. Figure 5a shows that MC3T3-E1 cells did express EGFR, and its phosphorylation was induced by EGF treatment. The inhibition of EGFR phosphorylation by erlotinib was also confirmed. EGF induced RANKL expression in MC3T3-E1 cells, and the expression was significantly suppressed by erlotinib in a dose dependent manner (Fig. 5b). Interestingly, the expression of OPG, the decoy receptor for RANKL, was not affected by either EGF or erlotinib treatment (Fig. 5c).
Discussion The present study demonstrated that erlotinib significantly suppressed bone and lung metastases, but not liver metastases, by SBC-5 cells that did not express EGFR. These findings suggest the importance of the host microenvironment in the
EGF
-
- -
-
- + + + + +
µ ) Erlotinib (µM)
0
0.125 0.25
0.5
1
0
0.125 0.25 0.5
1
Fig. 5 The effect of erlotinib on osteoblastic cell line MC3T3-E1 cells. Murine osteoblastic cell line MC3T3-E1 cells were differentiated into the mature state as described in ‘‘Materials and methods’’ section. a Differentiated cells were treated with EGF (10 ng/ml) for 10 min in the presence or absence of erlotinib. Expression of EGFR and phosphorylated EGFR (p-EGFR) was detected by western blotting. b, c Differentiated MC3T3-E1 cells were treated with EGF (10 ng/ml) in the presence or absence of various concentrations of erlotinib for 48 h. The cells were harvested, and the expression of b RANKL and c OPG was detected by RT-PCR. Bars show the SEM of the means of triplicate cultures. *P \ 0.01
treatment of lung cancer with erlotinib. The formation of distant metastasis involves several sequential steps, including tumor growth in the primary site, invasion into blood vessels, arrest in the capillaries, extravasation, invasion and growth in target organs [24, 25]. Molecular interactions between cancer cells and their microenvironments play important roles that allow the cancer cells to survive at metastatic sites, [24–27]. Therefore, the efficacy of therapeutic agents against cancer metastases depends on their mechanisms of action on tumor cells as well as the host microenvironments. There is accumulating evidence that the EGFR ligandEGFR axis plays a role in in bone biology and pathology. The expression and functionality of EGFR has been clearly
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demonstrated in bone marrow mesenchymal stem cells (BMMSCs) [6, 28], and osteoblasts themselves differentiate from mesenchymal stem cells [29]. In addition, EGFR ligands, such as EGF and amphiregulin, stimulate the proliferation of BMMSCs and osteoblasts [30–32], suggesting that EGFR ligands are mitogens for BMMSCs and osteoblasts. EGFR signaling also indirectly affects osteoclast formation, although osteoclasts do not express functional EGFR [16]. The role of EGFR in osteoclast formation suggests that the balance of RANKL and OPG (the decoy receptor for RANKL), which are expressed by osteoblasts, is associated with differentiation. The current study showed that the expression of RANKL, not OPG, in osteoblasts was stimulated by EGF, and this might enhance osteoclast activation via the RANK/RANKL axis. This result is supported by previous study reported by Furugaki et al. [33]. Using mouse osteolytic bone invasion model, they showed that the lung cancer cells induced RANKL expression of osteoblasts, and erlotinib inhibited the tumorinduced osteolytic invasion by suppressing osteoclast activation through inhibiting osteolytic factor production including RANKL. The importance of the balance of RANKL and OPG is also supported by Zhu et al. [16]. They demonstrated that EGFR ligands stimulate osteoclast formation by inhibiting the expression of OPG by osteoblasts, although RANKL expression was not affected. This difference might be due to differences in the experimental conditions and EGFR ligands. The importance of osteoclasts and osteoblasts on the development of bone metastases [16, 34] suggests that EGFR–TKIs prevent bone metastasis formation by inhibiting the activation of osteoclasts and osteoblasts in addition to their direct effect on tumor cells. Current evidence also suggests the association of EGFR signaling with primary bone tumor and bone metastasis formation. EGFR upregulation is observed in osteosarcomas and osteosarcoma cell lines [35], bone and soft tissue tumors [36]. Furthermore, EGFR is overexpressed in a variety of tumors metastasizing to bone. Prostate cancer is the best studied example. EGFR expression correlates with prostate cancer relapse and progression to androgen independence [37] and the blockade of EGFR signaling by PKI166, an EGFR–TKI, inhibits prostate cancer growth in the bones of nude mice [38]. Gefitinib, an EGFR–TKI, inhibits the recruitment of osteoclasts in metastatic bone lesions from breast cancer by affecting the ability of bone marrow stromal cells to induce osteoclast differentiation and activation [6]. Moreover, gefitinib administered to breast cancer patients (phase II trial) with bone metastasis significantly attenuates and relieves bone pain [6]. Collectively, EGFR–TKIs might thus have the potential to regulate the progression of bone metastases by acting on not only tumor cells but also host microenvironments, thus
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suggesting the importance of targeting the EGFR signaling blockade in the prevention of bone metastases. The current study found that erlotinib prevented distant metastases in the bone and the lungs but not in the liver, thus suggesting that organ heterogeneity is important in the pathogenesis of lung cancer metastasis. Organ heterogeneity in the therapeutic response is frequently observed in experimental xenograft models as well as in the clinical setting. Macrophage colony-stimulating factor gene transduction into human squamous cell lung carcinoma, RERF-LC-AI cells significantly suppresses experimental metastases in the liver but not the kidneys [13]. Treatment with, bevacizumab, an anti-human VEGF Ab, inhibits distant metastases of human ACC-LC-319/bone2 adenocarcinoma cells in the bone and the liver but not the lungs [14]. These observations imply that the heterogeneity of organ microenvironments affects the progression of lung cancer metastases. The current experiments did not reveal why erlotinib failed to inhibit liver metastases, but one plausible explanation is that hepatocyte growth factor (HGF), which is produced by stromal cells in the liver as well as other organs, might be responsible for organ heterogeneity in the therapeutic response observed in this study. HGF acts as a potent mitogen for endothelial cells and binds to receptors expressed on endothelial cells, thus stimulating angiogenesis [39]. Yano et al. [40] demonstrated that HGF induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations by restoring the phosphatidylinositol 3-kinase/Akt signaling pathway via phosphorylation of MET. These reports suggest that the microenvironment in the liver may not be favorable for EGFR–TKIs to exert antitumor activity. The current in vivo results also showed that erlotinib had therapeutic efficacy against lung metastases. Pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of breast cancer cells and facilitates lung metastasis, indicating that vascular normalization might lead to the suppression of lung metastasis [41]. In addition, Cerniglia et al. [7] reported that erlotinib treatment with tumor-bearing mice alters vessel morphology and decreases vessel permeability in tumor xenografts, resulting in vascular normalization. Although erlotinib failed to show an inhibitory effect on tumor angiogenesis in this study, these findings indicate that EGFR–TKIs might prevent lung metastases by decreasing vessel permeability and preventing extravasation of tumor cells. Baker et al. [42] have previously reported that the EGFR expression of host cells such as endothelial cells is conditioned by tumor microenvironment. Thus, there is a possibility that EGFR expression status of SBC-5 cells might also be regulated by host organ microenvironment, although it is still unclear that EGFR expression of cancer cells is conditioned by
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tumor microenvironment. Further studies are warranted to elucidate the mechanism of organ heterogeneity in the therapeutic response by erlotinib. In summary, the current study demonstrated that erlotinib significantly suppressed bone and lung metastases by SBC-5 cells which did not express EGFR, while it had no inhibitory effect on the proliferation of SBC-5 cells in vitro. Even though the precise mechanisms remain uncertain, these results strongly suggested that erlotinib prevented distant metastasis formation by affecting host microenvironments irrespective of its direct effect on tumor cells. Erlotinib might therefore be a promising therapeutic candidate for the inhibition of distant metastases. Acknowledgments This work was supported in part by a Grant-inaid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, and Ministry of Health and Welfare of Japan.
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11.
12.
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